Subtilase variants

ABSTRACT

The present invention relates to novel subtilase variants exhibiting improvements relative to the parent subtilase in one or more properties including: wash performance, thermal stability, storage stability or catalytic activity. The variants of the invention are suitable for use in e.g., cleaning or detergent compositions, such as laundry detergent compositions and dish wash compositions, including automatic dish wash compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority or the benefit under 35 U.S.C. 119 ofDanish application no. PA 2005 01007 filed Jul. 8, 2005 and U.S.provisional application No. 60/698,254 filed Jul. 11, 2005, the contentsof which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel subtilase variants exhibitingalterations relative to the parent subtilase in one or more propertiesincluding: wash performance, thermal stability, storage stability andcatalytic activity. The variants of the invention are suitable for usein e.g., cleaning or detergent compositions, such as laundry detergentcompositions and dish wash compositions, including automatic dish washcompositions. The present invention also relates to isolated DNAsequences encoding the variants, expression vectors, host cells, andmethods for producing and using the variants of the invention. Further,the present invention relates to cleaning and detergent compositionscomprising the variants of the invention.

BACKGROUND OF THE INVENTION

In the detergent industry enzymes have for more than 30 years beenimplemented in washing formulations. Enzymes used in such formulationscomprise proteases, lipases, amylases, cellulases, as well as otherenzymes, or mixtures thereof. Commercially the most important enzymesare proteases.

An increasing number of commercially used proteases are proteinengineered variants or naturally occurring wild type proteases, e.g.,RELASE®, ALCALASE®, SAVINASE®, PRIMASE®, EVERLASE®, ESPERASE®, OVOZYME®,CORONOASE, POLARZYME® and KANNASE® (Novozymes A/S), MAXATASE™, MAXACAL™,MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™, FN2™, FN3™, FN4™ andPURAFECT PRIME™ (Genencor International, Inc.), BLAP X and BLAP S(Henkel). Further, a number of protease variants are described in theart. A list of prior art protease variants is given in WO 99/27082.

However, even though a large number of useful protease variants havebeen described, there is still a need for new improved proteases orprotease variants for a number of industrial uses such as laundry orhard surface cleaning. Therefore, an object of the present invention isto provide improved subtilase variants for such purposes.

SUMMARY OF THE INVENTION

Thus, in a first aspect the present invention relates to a subtilasevariant comprising one or more of the modifications listed in Table 1.TABLE 1 Modifications in subtilase variants. T143K, Y167A, R170S, A194PY167A, R170S, A194P, K251R Y167A, R170S, A194P, S265K Y167A, R170S,A194P, V244R S141E, Y167A, R170S, A194P Y167A, R170S, M1751 Y167A,R170S, A172T Y167A, R170S, A174V, M175F Y167A, R170S, A172V, A174VY167A, R170S, A172E Y167A, R170S, M175L Y167A, R170S, A174T Y167A,R170S, A174T, M175L G53C, G61E A98S, S99D, G100S S9R, T22A, V68A, S99A,*99Ad S9R, P14H, R19L, N62D G61P, *99aS N43S, N62D *96aG, P131S, V203A,A228T N62D, A232C, Q236L, Q245N *96aA, A98T, R247K S99D, S101R, S103A,V104I, G160S, A194P, L217D *61aD N62D, S106A V68A, S106M, N184D S9R,A15T, *97aV, H120N A15M, A16P, *99aD *99aE, G160S, S163T, G195S, G211S,K237R, G258A, T260L G23S, *99aD, A194P, S242T, Q245R G100S, N173D Y167A,R170S, A172E A98T, Q137L, Y167A, R170S, M175L *98aA, S99D S99A, *99aD,V203A N62D, K237R V11M, N76D, L126F, K251R S9F, A15L, A16P, T22I, *98aA,S99D, R170H *96aA, *130aG, P131H E54D, N62D *98aA, *98bS, S99G, S101TS9R, A15T, V68A, I79T, G102S, P131H, Q137H *100aA, *100bG, *100cS,*100dG V68A, L111I *98aA, R170H, Q245R I35V, N62D, N183D, T224S *97aG,P131S, V203A, A228T S9R, R10K, P14Q, T22A, Y167A, R170S S9R, *22aL,S57A, G61E, *98aA, V139L, N173S P14T, N18K, Y167A, R170S S9R, Q12E,P14Q, K27R, Y167A, R170S N62D, R170L N62D, R170S, Q245R Y167A, R170S,A194P, K251R, S265K P14T, N18K, Y167A, R170S, A194P N62D, A151G, K237RN62D, A151G, Q245R N62D, A151G, K237R, Q245R S103A, V104I, G159D, A232V,Q236H, Q245R S9R, A15T, T22A, V139L S9R, A15T, G61E, A85T, E89Q, P239L,Q245C S9R, A15T, V68A, H120N, Q245R N248R S9R, A15T, *22AI, V139L,N204D, Q245L N218S S9R, A15T, V68A, Q245R, N252K S9R, A15T, V68A, Q245R,H120N V68A, S106A, H120N V68A, S106A, N252K A15T, V68A, S99G, Q245R,N261D S9R, V68A, S99G, Q245R, N261D V68A, S99G, Q245R, N261D S9R, A15T,V68A, S99G, N261D S9R, A15T, V68A, Q245R, N261D S9R, A15T, *22aL, V139L,S163G, N204D, Q245L Q245R, N252H S9R, *22aL, G61E, *97aA, M119I, Q137H,N173S V68A, S106A, T213A S9R, A15T, V68A, H120N, P131S, Q137H, Q245MS9R, A15T, V68A, I72F, S99G, Q245R, N261D S9R, A15T, V68A, S99D, Q245R,N261D S9R, A15T, V68A, S99G, A194P, Q245R, N261D S9R, A15T, V68A, N76I,S99G, Q245R, N261D S9R, A15T, V68A, S99G, A228V, Q245R, N261D

The variants listed in Table 1 exhibit protease activity. Each positioncorresponds to a position of the amino acid sequence of subtilisin BPN′set forth in FIG. 1 and SEQ ID NO: 1.

In a second aspect the present invention relates to an isolatedpolynucleotide encoding a subtilase variant of the invention.

In a third aspect the present invention relates to an expression vectorcomprising the isolated polynucleotide of the invention.

In a fourth aspect the present invention relates to a microbial hostcell transformed with the expression vector of the invention.

In a fifth aspect the present invention relates to a method forproducing a subtilase variant according to the invention, comprisingculturing a host according to the invention under conditions conduciveto the expression and secretion of the variant, and recovering thevariant.

In a sixth aspect the present invention relates to a cleaning ordetergent composition, preferably a laundry or dish wash composition,comprising the variant of the invention.

Concerning alignment and numbering, reference is made to FIG. 1 whichshows an alignment between subtilisin BPN′ (a) (BASBPN) and subtilisin309 (b) (BLSAVI). This alignment is in this patent application used as areference for numbering the residues.

Definitions

Prior to discussing this invention in further detail, the followingterms and conventions will first be defined. For a detailed descriptionof the nomenclature of amino acids and nucleic acids, we refer to WO00/71691 beginning at page 5, which is herein incorporated by reference.

Nomenclature and Conventions for Designation of Variants

In describing the various subtilase enzyme variants produced orcontemplated according to the invention, the following nomenclatures andconventions have been adapted for ease of reference: A frame ofreference is first defined by aligning the isolated or parent enzymewith subtilisin BPN′ (BASBPN).

The alignment can be obtained by the GAP routine of the GCG packageversion 9.1 to number the variants using the following parameters: gapcreation penalty=8 and gap extension penalty=8 and all other parameterskept at their default values.

Another method is to use known recognized alignments between subtilases,such as the alignment indicated in WO 91/00345. In most cases thedifferences will not be of any importance.

Thereby a number of deletions and insertions will be defined in relationto BASBPN (SEQ ID NO: 1). In FIG. 1, subtilisin 309 (SEQ ID NO: 2) has 6deletions in positions 36, 58, 158, 162, 163, and 164 in comparison toBASBPN. These deletions are in FIG. 1 indicated by asterixes (*). For adetailed description of the nomenclature of modifications introduced ina polypeptide by genetic manipulation we refer to WO 00/71691 page 7-12,which is herein incorporated by reference.

Proteases Enzymes cleaving the amide linkages in protein substrates areclassified as proteases, or (interchangeably) peptidases (see Walsh,1979, Enzymatic Reaction Mechanisms. W.H. Freeman and Company, SanFrancisco, Chapter 3).

Numbering of amino acid positions/residues If nothing else is mentionedthe amino acid numbering used herein correspond to that of the subtilaseBPN′ (BASBPN) sequence. For further description of the BPN′ sequence,see FIG. 1, SEQ ID NO: 1 or Siezen et al., 1991, Protein Engng.4:719-737.

Serine proteases A serine protease is an enzyme which catalyzes thehydrolysis of peptide bonds, and in which there is an essential serineresidue at the active site (White, Handler and Smith, 1973 “Principlesof Biochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp.271-272). The bacterial serine proteases have molecular weights in the20,000 to 45,000 Dalton range. They are inhibited bydiisopropylfluorophosphate. They hydrolyze simple terminal esters andare similar in activity to eukaryotic chymotrypsin, also a serineprotease. A more narrow term, alkaline protease, covering a sub-group,reflects the high pH optimum of some of the serine proteases, from pH9.0 to 11.0 (for review, see Priest, 1977, Bacteriological Rev.41:711-753).

Subtilases A sub-group of the serine proteases tentatively designatedsubtilases has been proposed by Siezen et al., 1991, Protein Engng.4:719-737 and Siezen et al., 1997, Protein Science 6:501-523. They aredefined by homology analysis of more than 170 amino acid sequences ofserine proteases previously referred to as subtilisin-like proteases. Asubtilisin was previously often defined as a serine protease produced byGram-positive bacteria or fungi, and according to Siezen et al., now isa subgroup of the subtilases. A wide variety of subtilases have beenidentified, and the amino acid sequence of a number of subtilases hasbeen determined. For a more detailed description of such subtilases andtheir amino acid sequences reference is made to Siezen et al. (1997).

One subgroup of the subtilases, I-S1 or “true” subtilisins, comprisesthe “classical” subtilisins, such as subtilisin 168 (BSS168), subtilisinBPN′, subtilisin Carlsberg (ALCALASE®, Novozymes A/S), and subtilisin DY(BSSDY).

A further subgroup of the subtilases, I-S2 or high alkaline subtilisins,is recognized by Siezen et al. (supra). Sub-group I-S2 proteases aredescribed as highly alkaline subtilisins and comprises enzymes such assubtilisin PB92 (BAALKP) (MAXACAL®, Genencor International Inc.),subtilisin 309 (SAVINASE®, Novozymes A/S), subtilisin 147 (BLS147)(ESPERASE®, Novozymes A/S), and alkaline elastase YaB (BSEYAB).

“SAVINASE®”. SAVINASE® which is marketed by Novozymes A/S is subtilisin309 from B. lentus and differs from BMLKP only in one position (N87S).SAVINASE® has the amino acid sequence designated b) in FIG. 1 and in SEQID NO: 2.

Parent subtilase. The term “parent subtilase” describes a subtilasedefined according to Siezen et al. (1991and 1997). For further detailssee description of “Subtilases” above. A parent subtilase may also be asubtilase isolated from a natural source, wherein subsequentmodifications have been made while retaining the characteristic of asubtilase. Furthermore, a parent subtilase may be a subtilase which hasbeen prepared by the DNA shuffling technique, such as described by J. E.Ness et al., 1999, Nature Biotechnology 17:893-896. Alternatively theterm “parent subtilase” may be termed “wild type subtilase”.

Modification(s) of a subtilase variant. The term “modification(s)” usedherein is defined to include chemical modification of a subtilase aswell as genetic manipulation of the DNA encoding a subtilase. Themodification(s) can be replacement(s) of the amino acid side chain(s),substitution(s), deletion(s) and/or insertions in or at the aminoacid(s) of interest.

Subtilase variant. In the context of this invention, the term subtilasevariant or mutated subtilase means a subtilase that has been produced byan organism which is expressing a mutant gene derived from a parentmicroorganism which possessed an original or parent gene and whichproduced a corresponding parent enzyme, the parent gene having beenmutated in order to produce the mutant gene from which said mutatedsubtilase protease is produced when expressed in a suitable host.

Homologous subtilase sequences. The homology between two amino acidsequences is in this context described by the parameter “identity”. Inorder to determine the degree of identity between two subtilases the GAProutine of the GCG package version 9.1 can be applied (infra) using thesame settings. The output from the routine is besides the amino acidalignment the calculation of the “Percent Identity” between the twosequences. Based on this description it is routine for a person skilledin the art to identify suitable homologous subtilases, which can bemodified according to the invention.

Isolated polynucleotide. The term “isolated”, when applied to apolynucleotide, denotes that the polynucleotide has been removed fromits natural genetic milieu and is thus free of other extraneous orunwanted coding sequences, and is in a form suitable for use withingenetically engineered protein production systems. Such isolatedmolecules are those that are separated from their natural environmentand include cDNA and genomic clones. Isolated DNA molecules of thepresent invention are free of other genes with which they are ordinarilyassociated, but may include naturally occurring 5′ and 3′ untranslatedregions such as promoters and terminators. The identification ofassociated regions will be evident to one of ordinary skill in the art(see for example, Dynan and Tijan, Nature 316:774-78, 1985). The term“an isolated polynucleotide” may alternatively be termed “a clonedpolynucleotide”.

Isolated protein. When applied to a protein, the term “isolated”indicates that the protein has been removed from its native environment.In a preferred form, the isolated protein is substantially free of otherproteins, particularly other homologous proteins (i.e., “homologousimpurities” (see below)). An isolated protein is more than 10% pure,preferably more than 20% pure, more preferably more than 30% pure, asdetermined by SDS-PAGE. Further it is preferred to provide the proteinin a highly purified form, i.e., more than 40% pure, more than 60% pure,more than 80% pure, more preferably more than 95% pure, and mostpreferably more than 99% pure, as determined by SDS-PAGE. The term“isolated protein” may alternatively be termed “purified protein”.

Homologous impurities. The term “homologous impurities” means anyimpurity (e.g., another polypeptide than the subtilase of theinvention), which originate from the homologous cell where the subtilaseof the invention is originally obtained from.

Obtained from. The term “obtained from” as used herein in connectionwith a specific microbial source, means that the polynucleotide and/orsubtilase produced by the specific source, or by a cell in which a genefrom the source has been inserted.

Substrate. The term “substrate” used in connection with a substrate fora protease should be interpreted in its broadest form as comprising acompound containing at least one peptide (amide) bond susceptible tohydrolysis by a subtilisin protease.

Product. The term “product” used in connection with a product derivedfrom a protease enzymatic reaction should, in the context of the presentinvention, be interpreted to include the products of a hydrolysisreaction involving a subtilase protease. A product may be the substratein a subsequent hydrolysis reaction.

Wash Performance. In the present context the term “wash performance” isused as an enzyme's ability to remove proteinaceous or organic stainspresent on the object to be cleaned during e.g., wash or hard surfacecleaning. See also the wash performance test in Example 3 herein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an alignment between subtilisin BPN′ (a) and SAVINASE® (b)using the GAP routine mentioned above.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel subtilase variants exhibitingalterations relative to the parent subtilase in one or more propertiesincluding: Wash performance, thermal stability, storage stability orcatalytic activity. Variants which are contemplated as being part of theinvention are such variants where, when compared to the wild-typesubtilase, one or more amino acid residues have been modified bysubstitution, deletion or insertion. The variants of the presentinvention comprise one or more of those modifications listed in Table 1.

The variants listed in Table 1 exhibit protease activity, and eachposition corresponds to a position of the amino acid sequence ofsubtilisin BPN′ showed in FIG. 1 and SEQ ID NO: 1.

A subtilase variant of the first aspect of the invention may be a parentor wild-type subtilase identified and isolated from nature. Such aparent wild-type subtilase may be specifically screened for by standardtechniques known in the art.

One preferred way of doing this may be by specifically PCR amplifyconserved DNA regions of interest from subtilases from numerousdifferent microorganism, preferably different Bacillus strains.

Subtilases are a group of conserved enzymes, in the sense that their DNAand amino acid sequences are homologous. Accordingly it is possible toconstruct relatively specific primers flanking the polynucleotidesequences of interest.

Using such PCR primers to amplify DNA from a number of differentmicroorganisms, preferably different Bacillus strains, followed by DNAsequencing of said amplified PCR fragments, it will be possible toidentify strains which produce subtilase variants of the invention.Having identified the strain and a partial DNA sequence of such asubtilase of interest, it is routine work for a person skilled in theart to complete cloning, expression and purification of such asubtilase. However, it is envisaged that a subtilase variant of theinvention is predominantly a variant of a parent subtilase.

A subtilase variant suitable for the uses described herein may beconstructed by standard techniques known in the art such as bysite-directed/random mutagenesis or by DNA shuffling of differentsubtilase sequences. See the “Material and Methods” section and Example1 herein for further details.

As will be acknowledged by the skilled person, the variants describedherein may comprise one or more additional modifications, in particularone or more additional substitutions or insertions. Moreover, thevariants described herein may encompass mutation at more than just oneposition. For example the variant according to the invention may containmutations at one position, two positions, three positions or more thanthree positions, such as four to eight positions. It is preferred thatthe parent subtilase belongs to the subgroups I-S1 or I-S2, especiallysubgroup I-S2, both for enzymes from nature or from the artificialcreation of diversity, and for designing and producing variants from aparent subtilase.

In relation to variants from subgroup I-S1, it is preferred to select aparent subtilase from the group consisting of BSS168 (BSSAS, BSAPRJ,BSAPRN, BMSAMP), BASBPN, BSSDY, BLSCAR (BLKERA, BLSCA1, BLSCA2, BLSCA3),BSSPRC, and BSSPRD, or functional variants thereof having retained thecharacteristic of sub-group I-S1.

In relation to variants from subgroup I-S2 it is preferred to select aparent subtilase from the group consisting of BSAPRQ, BLS147 (BSAPRM,BAH101), BLSAVI (BSKSMK, BAALKP, BLSUBL), BYSYAB, BAPB92, TVTHER, andBSAPRS, or functional variants thereof having retained thecharacteristic of sub-group I-S2. In particular, the parent subtilase isBLSAVI (SAVINASE®, Novozymes A/S), and a preferred subtilase variant ofthe invention is accordingly a variant of SAVINASE®.

The present invention also encompasses any of the above mentionedsubtilase variants in combination with any other modification to theamino acid sequence thereof. Especially combinations with othermodifications known in the art to provide improved properties to theenzyme are envisaged. The art describes a number of subtilase variantswith different improved properties and a number of those are mentionedin the “Background of the invention” section. Those references aredisclosed here as references to identify a subtilase variant, whichadvantageously can be combined with a subtilase variant describedherein. Such combinations comprise the positions: 222 (improvesoxidation stability), 218 (improves thermal stability), substitutions inthe Ca²⁺-binding sites stabilizing the enzyme, e.g., position 76, andmany other apparent from the prior art.

In further embodiments a subtilase variant described herein mayadvantageously be combined with one or more modification(s) in any ofthe positions:

27, 36, 56, 76, 87, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 120,123, 159, 167, 170, 206, 218, 222, 224, 232, 235, 236, 245, 248, 252 and274.

Specifically, the following BLSAVI, BLSUBL, BSKSMK, and BAALKPmodifications are considered appropriate for combination:

K27R, *36D, S56P, N62D, V68A, N76D, S87N, G97N, S99SE, S101G, S101R,S103A, V104A, V104I, V104N, V104Y, S106A, H120D, H120N, N123S, G159D,Y167A, R170S, R170L, A194P, N204D, V2051, Q206E, L217D, N218S, N218D,M222S, M222A, T224S, A232V, K235L, Q236H, Q245R, N248D, N252K and T274A.

Furthermore variants comprising any of the modifications S101G+V104N,S87N+S100G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I,S99D+S101R+S103A+V104I+G160S,S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D,S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D,S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I or N76D+V104A, or othercombinations of the modifications K27R, *36D, S56P, N62D, V68A, N76D,S87N, G97N, S99SE, S101G, S103A, V104A, V104I, V104N, V104Y, S106A,H120D, H120N, N123S, G159D, Y167A, R170S, R170L, A194P, N204D, V2051,Q206E, L217D, N218D, N218S, M222A, M222S, T224S, A232V, K235L, Q236H,Q245R, N248D, N252K and T274A in combination with any one or more of themodification(s) mentioned above exhibit improved properties. Aparticular interesting variant is a variant, which, in addition to amodification according to the invention, contains the followingsubstitutions: S101 G+S103A+V104I+G159D+A232V+Q236H+ Q245R+N248D+N252K.

Moreover, subtilase variants of the main aspect(s) of the invention arepreferably combined with one or more modification(s) in any of thepositions 129, 131and 194, preferably as 129K, 131H and 194Pmodifications, and most preferably as P129K, P131H and A194Pmodifications. Any of those modification(s) are expected to provide ahigher expression level of the subtilase variant in the productionthereof.

The wash performance of a selected variant of the invention may betested in the wash performance test disclosed in Example 3 herein. Thewash performance test may be employed to assess the ability of avariant, when incorporated in a standard or commercial detergentcomposition, to remove proteinaceous stains from a standard textile ascompared to a reference system, namely the parent subtilase or a similarsubtilase exhibiting an even better wash performance (incorporated inthe same detergent system and tested under identical conditions). Theenzyme variants of the present application were tested using theAutomatic Mechanical Stress Assay (AMSA). With the AMSA test the washperformance of a large quantity of small volume enzyme-detergentsolutions can be examined rapidly. Using this test, the wash performanceof a selected variant can be initially investigated, the rationale beingthat if a selected variant does not show a significant improvement inthe test compared to the parent subtilase, it is normally not necessaryto carry out further test experiments.

Therefore, variants which are particularly interesting for the purposesdescribed herein, are such variants which, when tested in a commercialdetergent composition such as a US type detergent, an Asian type, aEuropean type or a Latin American type detergent as described in thewash performance test (Example 3), shows an improved wash performance ascompared to the parent subtilase tested under identical conditions.

The improvement in the wash performance may be quantified by calculatingthe so-called intensity value (Int) defined in Example 3, herein.

Evidently, it is preferred that the variant of the invention fulfils theabove criteria on at least the stated lowest level, more preferably atthe stated highest level.

Producing a Subtilase Variant

Many methods for cloning a subtilase and for introducing substitutions,deletions or insertions into genes (e.g., subtilase genes) are wellknown in the art.

In general standard procedures for cloning of genes and introducingmutations (random and/or site directed) into said genes may be used inorder to obtain a subtilase variant of the invention. For furtherdescription of suitable techniques reference is made to Example 1 herein(vide infra) and (Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel etal. (eds.) “Current protocols in Molecular Biology”. John Wiley andSons, 1995; Harwood and Cutting (eds.) “Molecular Biological Methods forBacillus”. John Wiley and Sons, 1990), and WO 96/34946.

Further, a subtilase variant may be constructed by standard techniquesfor artificial creation of diversity, such as by DNA shuffling ofdifferent subtilase genes (WO 95/22625; Stemmer, 1994, Nature370:389-91). DNA shuffling of, e.g., the gene encoding SAVINASE® withone or more partial subtilase sequences identified in nature, will aftersubsequent screening for improved wash performance variants, providesubtilase variants suitable for the purposes described herein.

Expression Vectors

A recombinant expression vector comprising a DNA construct encoding theenzyme of the invention may be any vector that may conveniently besubjected to recombinant DNA procedures. The choice of vector will oftendepend on the host cell into which it is to be introduced. Thus, thevector may be an autonomously replicating vector, i.e., a vector thatexists as an extra-chromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid.

Alternatively, the vector may be one that on introduction into a hostcell is integrated into the host cell genome in part or in its entiretyand replicated together with the chromosome(s) into which it has beenintegrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the enzyme of the invention is operably linked to additionalsegments required for transcription of the DNA. In general, theexpression vector is derived from plasmid or viral DNA, or may containelements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g., transcription initiates in a promoter andproceeds through the DNA sequence coding for the enzyme.

The promoter may be any DNA sequence that shows transcriptional activityin the host cell of choice and may be derived from genes encodingproteins either homologous or heterologous to the host cell.

Examples of suitable promoters for use in bacterial host cells includethe promoter of the Bacillus stearothermophilus maltogenic amylase gene,the Bacillus licheniformis alpha-amylase gene, the Bacillusamyloliquefaciens alpha-amylase gene, the Bacillus subtilis alkalineprotease gene, or the Bacillus pumilus xylosidase gene, or the phageLambda P_(R) or P_(L) promoters or the E. coli lac, trp or tacpromoters. The DNA sequence encoding the enzyme of the invention mayalso, if necessary, be operably connected to a suitable terminator.

The recombinant vector of the invention may further comprise a DNAsequence enabling the vector to replicate in the host cell in question.The vector may also comprise a selectable marker, e.g., a gene theproduct of which complements a defect in the host cell, or a geneencoding resistance to e.g., antibiotics like kanamycin,chloramphenicol, erythromycin, tetracycline, spectinomycine, or thelike, or resistance to heavy metals or herbicides.

To direct an enzyme of the present invention into the secretory pathwayof the host cells, a secretory signal sequence (also known as a leadersequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the enzyme in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe enzyme. The secretory signal sequence may be that normallyassociated with the enzyme or may be from a gene encoding anothersecreted protein.

The procedures used to ligate the DNA sequences coding for the presentenzyme, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, or to assemble these sequences bysuitable PCR amplification schemes, and to insert them into suitablevectors containing the information necessary for replication orintegration, are well known to persons skilled in the art (cf., forinstance, Sambrook et al., op. cit.).

Host Cell

The DNA sequence encoding the present enzyme introduced into the hostcell may be either homologous or heterologous to the host in question.If homologous to the host cell, i.e., produced by the host cell innature, it will typically be operably connected to another promotersequence or, if applicable, another secretory signal sequence and/orterminator sequence than in its natural environment. The term“homologous” is intended to include a DNA sequence encoding an enzymenative to the host organism in question. The term “heterologous” isintended to include a DNA sequence not expressed by the host cell innature. Thus, the DNA sequence may be from another organism, or it maybe a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector ofthe invention is introduced may be any cell that is capable of producingthe present enzyme and includes bacteria, yeast, fungi and highereukaryotic cells including plants.

Examples of bacterial host cells which, on cultivation, are capable ofproducing the enzyme of the invention are gram-positive bacteria such asstrains of Bacillus, such as strains of B. subtilis, B. licheniformis,B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B.amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megateriumor B. thuringiensis, or strains of Streptomyces, such as S. lividans orS. murinus, or gram-negative bacteria such as Escherichia coli.

The transformation of the bacteria may be effected by protoplasttransformation, electroporation, conjugation, or by using competentcells in a manner known per se (cf. Sambrook et al., supra).

When expressing the enzyme in bacteria such as E. coli, the enzyme maybe retained in the cytoplasm, typically as insoluble granules (known asinclusion bodies), or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysedand the granules are recovered and denatured after which the enzyme isrefolded by diluting the denaturing agent. In the latter case, theenzyme may be recovered from the periplasmic space by disrupting thecells, e.g., by sonication or osmotic shock, to release the contents ofthe periplasmic space and recovering the enzyme.

When expressing the enzyme in gram-positive bacteria such as Bacillus orStreptomyces strains, the enzyme may be retained in the cytoplasm, ormay be directed to the extracellular medium by a bacterial secretionsequence. In the latter case, the enzyme may be recovered from themedium as described below.

Method for Producing a Subtilase Variant

The present invention provides a method of producing an isolated enzymeaccording to the invention, wherein a suitable host cell, which has beentransformed with a DNA sequence encoding the enzyme, is cultured underconditions permitting the production of the enzyme, and the resultingenzyme is recovered from the culture.

When an expression vector comprising a DNA sequence encoding the enzymeis trans-formed into a heterologous host cell it is possible to enableheterologous recombinant production of the enzyme of the invention.Thereby it is possible to make a highly purified subtilase composition,characterized in being free from homologous impurities.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase may conveniently be secreted into the culture mediumand may be recovered there-from by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulfate, followed by chromatographic procedures such asion exchange chromatography, affinity chromatography, or the like.

Detergent Applications

The enzyme of the invention may be added to and thus become a componentof a detergent composition. The detergent composition of the inventionmay for example be formulated as a hand or machine laundry detergentcomposition including a laundry additive composition suitable forpre-treatment of stained fabrics and a rinse added fabric softenercomposition, or be formulated as a detergent composition for use ingeneral household hard surface cleaning operations, or be formulated forhand or machine dishwashing operations.

In a specific aspect, the invention provides a detergent additivecomprising the enzyme of the invention. The detergent additive as wellas the detergent composition may comprise one or more other enzymes suchas a protease, a lipase, a cutinase, an amylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a laccase, and/or a peroxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metallo protease, preferably an alkaline microbialprotease or a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 68,76, 87, 97, 101, 104, 106, 120, 123, 167, 170, 194, 206, 218, 222, 224,235, 245, 252 and 274. Preferred commercially used protease enzymesinclude RELASE®, ALCALASE®, SAVINASE®, PRIMASE®, EVERLASE®, ESPERASE®,OVOZYME®, CORONASE®, POLARZYME® and KANNASE® (Novozymes A/S), MAXATASE™,MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™, FN2™, FN3™,FN4™ and PURAFECT PRIME™ (Genencor International, Inc.), BLAP X and BLAPS (Henkel).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta1131:253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422). Other examples are lipase variants such as those described inWO 92/05249, WO 94/01541, EP 407225, EP 260105, WO 95/35381, WO96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO97/04079 and WO 97/07202. Preferred commercially used lipase enzymesinclude LIPOLASE®, LIPOLASE ULTRA® and LIPEX® (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin.

Chemically modified or protein engineered mutants are included. Amylasesinclude, for example, α-amylases obtained from Bacillus, e.g., a specialstrain of B. licheniformis, described in more detail in GB 1,296,839.Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444. Commercially used amylases areDURAMYL®, TERMAMYL®, STAINZYME®, FUNGAMYL® and BAN® (Novozymes A/S),RAPIDASE™, PURASTAR™ and PURASTAR OXAM™ (from Genencor InternationalInc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungalcellulases produced from Humicola insolens, Myceliophthora thermophilaand Fusarium oxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO89/09259. Especially suitable cellulases are the alkaline or neutralcellulases having colour care and whiteness maintenance benefits.Examples of such cellulases are cellulases described in EP 0 495 257, EP0 531 372, WO 96/11262, WO 96/29397, WO 98/08940. Other examples arecellulase variants such as those described in WO 94/07998, EP 0 531 315,U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,686,593, U.S. Pat. No.5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299. Commerciallyused cellulases include RENOZYME®, CELLUZYME®, and CAREZYME® (NovozymesA/S), CLAZINASE™, and PURADEX HA™ (Genencor Int. Inc.), and KAC-500(B)™(Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.Commercially used peroxidases include GUARDZYME™ (Novozymes A/S).

Hemicellulases: Suitable hemicellulases include those of bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Suitable hemicellulases include mannanase, lichenase,xylanase, arabinase, galactanase acetyl xylan esterase, glucorunidase,ferulic acid esterase, coumaric acid esterase and arabinofuranosidase asdescribed in WO 95/35362. Suitable mannanases are described in WO99/64619.

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated e.g., as a granulate, a liquid, a slurry, etc. Preferreddetergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethylene glycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste, a gel or aliquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpoly-glycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethyl-cellulose, poly(vinylpyrrolidone), poly (ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymersand lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of e.g., the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in e.g., WO 92/19709and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

It is at present contemplated that in the detergent compositions anyenzyme, in particular the enzyme of the invention, may be added in anamount corresponding to 0.01-100 mg of enzyme protein per litre of washliquor, preferably 0.05-5 mg of enzyme protein per litre of wash liquor,in particular 0.1-1 mg of enzyme protein per litre of wash liquor.

Variations in local and regional conditions, such as water hardness andwash temperature calls for regional detergent compositions. DetergentExamples 1and 2 provide ranges for the composition of a typical LatinAmerican detergent and a typical European powder detergent respectively.

DETERGENT EXAMPLE 1 Typical Latin American Detergent Composition

Group Subname Content Surfactants 0-30% Sulphonates 0-30% Sulphates 0-5%Soaps 0-5% Non-ionics 0-5% Cationics 0-5% FAGA 0-5% Bleach 0-20% SPT/SPM0-15% NOBS, TAED 0-5% Builders 0-60% Phosphates 0-30% Zeolite 0-5%Na2OSiO2 0-10% Na2CO3 0-20% Fillers 0-40% Na2SO4 0-40% Others up to 100%Polymers Enzymes Foam regulators Water Hydrotropes Others

DETERGENT EXAMPLE 2 Typical European powder Detergent Composition

Group Subname Content Surfactants 0-30% Sulphonates 0-20% Sulphates0-15% Soaps 0-10% Non-ionics 0-10% Cationics 0-10% Other 0-10% Bleach0-30% SPT/SPM 0-30% NOBS + TAED 0-10% Builders 0-60% Phosphates 0-40%Zeolite 0-40% Na2OSiO2 0-20% Na2CO3 0-20% Fillers 0-40% Na2SO4 0-40%NaCl 0-40% Others up to 100% Polymers Enzymes Foam regulators WaterHydrotropes OthersOther Applications

The subtilase variants of the present invention may be used in theprocessing of food, especially in the field of diary products, such asmilk, cream and cheese, but also in the processing of meat andvegetables. The subtilase variants of the present invention may also beused in the processing of feed for cattle, poultry, and pigs andespecially for pet food. Further, the subtilase variants of theinvention may be used for the treatment of hides. The subtilase variantsof the invention may also be used in processes for decontaminatinginstruments, surfaces, and other materials in hospitals, clinics, andmeat processing plants, etc. in order to decompose prions or otherinfectious agents.

Materials and Methods

Method for Producing a Protease Variant

The present invention provides a method of producing an isolated enzymeaccording to the invention, wherein a suitable host cell, which has beentransformed with a DNA sequence encoding the enzyme, is cultured underconditions permitting the production of the enzyme, and the resultingenzyme is recovered from the culture.

When an expression vector comprising a DNA sequence encoding the enzymeis transformed into a heterologous host cell it is possible to enableheterologous recombinant production of the enzyme of the invention.Thereby it is possible to make a highly purified RP-II proteasecomposition, characterized in being free from homologous impurities.

The medium used to culture the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed subtilase variant may conveniently be secreted into theculture medium and may be recovered there-from by well-known proceduresincluding separating the cells from the medium by centrifugation orfiltration, precipitating proteinaceous components of the medium bymeans of a salt such as ammonium sulfate, followed by chromatographicprocedures such as ion exchange chromatography, affinity chromatography,or the like.

Proteolytic Activity

Enzyme activity can be measured using the PNA assay usingsuccinyl-alanine-alanine-proline-glutamicacid-paranitroaniline as asubstrate. The principle of the PNA assay is described in the Journal ofAmerican Oil Chemists Society, Rothgeb, T. M., Goodlander, B. D.,Garrison, P. H., and Smith, L. A., (1988).

Textiles

Standard textile pieces are obtained from EMPA St. Gallen,Lerchfeldstrasse 5, CH-9014 St. Gallen, Switzerland or CFT, Center ForTestmaterials, Vlaardingen, Netherlands. Especially important are EMPA116 (cotton textile stained with blood, milk and ink), EMPA 117(polyester/cotton textile stained with blood, milk and ink), C-03(cotton textile stained with chocolate milk and soot), C-05 (cottontextile stained with blood, milk and ink) and C-10 (cotton textilestained with milk, oil and pigment).

Wash Conditions Latin North Asia excl. Region America America EuropeJapan Japan Temperature 20-25° C. 20-32° C. 30-60° C. 15-30° C. 15-20°C. Washing 14-16 min 12 min 20-40 min 14-20 min 15 min time Water6-12°dH 6°dH 15°dH 14°dH 3°dH hardness* Detergent 1.5-4 g/l 1.0-1.5 g/l4-10 g/l 1.5-2.5 g/l 0.5-0.7 g/l dosage Washing pH As it is As it is Asit is As it is As it is*°dH: adjusted by adding CaCl₂*2H₂O, MgCl₂*6H₂O and NaHCO₃ to Milli-Qwater.Detergents

The enzymes of the invention may be tested in the detergent formulationsdisclosed in WO 97/07202 or in the detergent examples above. Further,tests could be done in detergents formulations purchased from wfktestgewebe GmbH (Germany) or similar supplier, or in commercialdetergents.

List of test detergents from wfk testgewebe:

IEC 60456 Type A* Base Detergent

IEC 60456 Type B Base Detergent

IEC 60456 Type C Detergent

ECE Reference Detergent with Phosphate (1977)

ECE Reference Detergent without Phosphate (1998)

AHAM Standard Detergent

EU ECOLABEL (detergents) Light Duty Detergent

EU ECOLABEL (detergents) PVP

However, also one of the following commercial detergents may be used inthe wash assay, e.g., Ariel HDP, P&G, Mexico; Omo Multi Acao HDP,Unilever, Brazil; Breeze HDP, Unilever Thailand; Diao Pai, Nice, China;Tide HDL, P&G, US; Wisk HDL, Unilever, US; TOP HDP, Lion, Japan; AttackHDP, Kao, Japan; Ariel Regular HDP, P&G, Europe; Ariel Compact HDPC,P&G, Europe; Persil Megaperls, Henkel, Germany and Persil, Unilever, UK.

Furthermore, a brand extension or color/compact version for the abovespecified detergent could be used as well.

If the detergent contains enzymes, the detergent should be in-activatedbefore use in order to eliminate the enzyme activity already present inthe detergent. This is done by heating a detergent stock solution to 85°C. in 5 minutes in a micro wave oven. The concentration of the detergentstock solution to be inactivated in the micro wave oven is 4-20 g/l.

Automatic Mechanical Stress Assay

The Automatic Mechanical Stress Assay (AMSA) is described in Example 3below.

Mini Wash Assay

The milliliter scale wash performance assay is conducted under thefollowing conditions: Detergent Latin American HDP Detergent dose 1.5-4g/l pH As it is Wash time 14-16 min. Temperature 20-25° C. Waterhardness 6-12°dH, adjusted by adding CaCl₂*2H₂O, MgCl₂*6H₂O and NaHCO₃to milli-Q water. Enzyme conc. 5 nM, 10 nM, 30 nM Test system 125 mlglass beakers. Textile dipped in test solution. Continuously lifted upand down into the detergent solution, 50 times per minute. Test solutionvolume 50 ml

After washing the textile piece is flushed in tap water and air-driedand the remission (R) of the test material is measured at 460 nm using aZeiss MCS 521 VIS spectrophotometer. The measurements are done accordingto the manufacturer's protocol.

The performance of the new variants is compared to the performance ofSavinase by calculating the relative performance:RP=(R_(variant) −R _(BLANK))/(R _(SAVINASE) −R _(BLANK))

A variant is considered to exhibit improved wash performance, if itperforms better than the reference in at least one detergentcomposition.

EXAMPLE 1

Construction and Expression of Enzyme Variants:

Site-Directed Mutagenesis:

Subtilisin 309 (SAVINASE®) site-directed variants of the inventioncomprising specific insertions/deletions/substitutions are made bytraditional cloning of DNA fragments (Sambrook et al., MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor, 1989)produced by PCR with oligonucleotides containing the desired mutations.

Briefly, plasmid DNA pSX222 (E. coli/B. subtilis shuttle vectorincluding appropriate selection marker, origins of replication forBacillus and E. coli, digestion sites, etc. disclosed in WO 96/34946)bearing the subtilisin 309 wild-type or a subtilisin 309 variant gene isused as template in the PCR reaction. In a first PCR an oligonucleotidecontaining the desired mutation (anti-sense) and a suitable oppositeoligonucleotide (sense) is used. The resulting DNA fragment is used as asense oligonucleotide in a second PCR together with a suitableanti-sense oligonucleotide. The resulting DNA fragment is digested withsuitable restriction enzymes and ligated into a suitable E.coli/Bacillus shuttle vector (e.g., pSX222) digested with the sameenzymes.

The ligation product is transformed into competent E. coli and plated ona solid agar containing an appropriate selection marker. DNA purifiedfrom a single colony is sequenced to confirm the designed mutation.Plasmid DNA is isolated from E. coli cells bearing plasmids containingsubtilisin 309 genes with the designed mutation and is transformed intoa suitable competent B. subtilis strain, i, B. subtilis DN1885:Disclosed in WO 01/16285 (e.g., as described by Dubnau et al., 1971, J.Mol. Biol. 56:209-221) and plated on a solid agar containing anappropriate selection marker.

Plasmid DNA from single Bacillus colonies showing protease activity isisolated and sequenced to confirm the designed mutation. Bacilluscolonies bearing plasmid DNA including subtilisin 309 genes with thedesired mutations are fermented in baffled shake flasks in a suitablemedia.

EXAMPLE 2

Purification and Assessment of Enzyme Concentration

After fermentation purification of subtilisin variants is accomplishedusing Hydrophobic Charge Induction Chromatography (HCIC) and subsequentvacuum filtration. To capture the enzyme, the HCIC uses a cellulosematrix to which 4-Mercapto-Ethyl-Pyridine (4-MEP) is bound.

Beads of the cellulose matrix sized 80-100 micro-m are mixed with amedia containing yeast extract and the transformed B. subtilis capableof secreting the subtilisin variants and incubated at pH 9.5 inUnifilter® microplates. As 4-MEP is hydrophobic at pH >7 and thesubtilisin variants are hydrophobic at pH 9.5 a hydrophobic associationis made between the secreted enzyme and the 4-MEP on the beads. Afterincubation the media and cell debris is removed by vacuum filtrationwhile the beads and enzyme are kept on the filter. To elute the enzymefrom the beads the pH is now lowered by washing the filter with anelution buffer (pH 5). Hereby the enzymes part from the beads and can beretrieved from the buffer.

The concentration of the purified subtilisin enzyme variants is assessedby active site titration (AST). The purified enzyme is incubated withthe high affinity inhibitor CI-2A at different concentrations to inhibita varying amount of the active sites. The protease and inhibitor bindsto each other at a 1:1 ratio and accordingly the enzyme concentrationcan be directly related to the concentration of inhibitor, at which allprotease is inactive. To measure the residual protease activity, asubstrate (0.6 mM Suc-Ala-Ala-Pro-Phe-pNA in Tris/HCl buffer) is addedafter the incubation with inhibitor and during the following 4 minutesthe development of the degradation product pNA (paranitrophenol) ismeasured periodically at 405 nm on an Elisa Reader. Each of the variantsof the invention listed in Table 1 herein was purified according to theabove procedure and subsequently the enzyme concentration wasdetermined.

Known concentrations of the variants of Table 1 were tested for washperformance in detergent as described below.

EXAMPLE 3

Wash Performance of Detergent Composition Comprising Subtilase Variants

The enzyme variants of the present application are tested using theAutomatic Mechanical Stress Assay (AMSA). With the AMSA test the washperformance of a large quantity of small volume enzyme-detergentsolutions can be examined. The AMSA plate has a number of slots for testsolutions and a lid firmly squeezing the textile swatch to be washedagainst all the slot openings. During the washing time, the plate, testsolutions, textile and lid are vigorously shaken to bring the testsolution in contact with the textile and apply mechanical stress. Forfurther description see WO 02/42740 especially the paragraph “Specialmethod embodiments” at page 23-24. The assay is conducted under theexperimental conditions specified below. Detergent Latin American typeHDP Detergent dosage 2.2 g/l Test solution volume 160 micro l pHAdjusted to pH 9.5-10.5 with NaHCO₃. Wash time 14 minutes Temperature20° C. Water hardness 9°dH* Enzyme concentration 5 nM, 10 nM and 30 nMin test solution Test material C-10*°dH: adjusted by adding CaCl₂*2H₂O; MgCl₂*6H₂O (Ratio Ca²⁺:Mg²⁺⁻ = 2:1)to milli-Q water.

The Latin American type detergent was composed according to theprovisions in Detergent Example 1 herein. After washing the textilepieces are flushed in tap water and air-dried.

The performance of the enzyme variant is measured as the brightness ofthe colour of the textile samples washed with that specific enzymevariant. Brightness can also be expressed as the intensity of the lightreflected from the textile sample when aluminated with white light. Whenthe textile is stained the intensity of the reflected light is lower,than that of a clean textile. Therefore the intensity of the reflectedlight can be used to measure wash performance of an enzyme variant.

Color measurements are made with a professional flatbed scanner (PFUDL2400pro), which is used to capture an image of the washed textilesamples. The scans are made with a resolution of 200 dpi and with anoutput colour dept of 24 bits. In order to get accurate results, thescanner is frequently calibrated with a Kodak reflective IT8 target. Toextract a value for the light intensity from the scanned images, aspecial designed software application is used (Novozymes Color VectorAnalyzer). The program retrieves the 24 bit pixel values from the imageand converts them into values for red (r), green (g) and blue (b). Theintensity value (Int) is calculated by adding the (r), (g) and (b)values together as vectors and then taking the length of the resultingvector:Int√{square root over (r ² +g ² +b ²)}.

The wash performance (P) of a variant is defined as the light intensityvalue of textile surface washed with enzyme variant:P=Int(v)

The results are presented in Table 2 where the performance is given asrelative performance of a new variant versus the performance of Savinaseat 10 nM protease concentration:RP is (P _(VARIANT) −P _(BLANK))/(P _(SAVINASE) −P _(BLANK)) TABLE 2Wash performance test results with subtilase variants relative to theperformance of Savinase. Relative Mutations in variant performanceT143K, Y167A, R170S, A194P 1.8 Y167A, R170S, A194P, K251R 1.6 Y167A,R170S, A194P, S265K 2.0 Y167A, R170S, A194P, V244R 1.9 S141E, Y167A,R170S, A194P 1.1 Y167A, R170S, M175I 1.2 Y167A, R170S, A172T 1.2 Y167A,R170S, A174V, M175F 1.4 Y167A, R170S, A172V, A174V 1.5 Y167A, R170S,A172E 1.3 Y167A, R170S, M175L 1.5 Y167A, R170S, A174T 1.2 Y167A, R170S,A174T, M175L 1.4 G53C, G61E 1.3 A98S, S99D, G100S 1.4 S9R, T22A, V68A,S99A, *99aD 1.7 S9R, P14H, R19L, N62D 1.8 G61P, *99aS 1.7 N43S, N62D 1.7*96aG, P131S, V203A, A228T 1.8 N62D, A232C, Q236L, Q245N 2.0 *96aA,A98T, R247K 2.0 S99D, S101R, S103A, V104I, G160S, A194P, L217D 1.4 *61aD1.3 N62D, S106A 2.2 V68A, S106M, N184D 1.6 S9R, A15T, *97aV, H120N 1.4A15M, A16P, *99aD 1.6 *99aE, G160S, S163T, G195S, G211S, K237R, 1.2G258A, T260L G23S, *99aD, A194P, S242T, Q245R 1.5 G100S, N173D 1.4Y167A, R170S, A172E 1.1 A98T, Q137L, Y167A, R170S, M175L 1.1 *98aA, S99D1.8 S99A, *99aD, V203A 1.8 N62D, K237R 2.1 V11M, N76D, L126F, K251R 1.4S9F, A15L, A16P, T22I, *98aA, S99D, R170H 1.2 *96aA, *130aG, P131H 1.5E54D, N62D 2.0 *98aA, *98bS, S99G, S101T 1.8 S9R, A15T, V68A, I79T,G102S, P131H, Q137H 1.7 *100aA, *100bG, *100cS, *100dG 1.7 V68A, L111I1.8 *98aA, R170H, Q245R 1.8 I35V, N62D, N183D, T224S 1.2 *97aG, P131S,V203A, A228T 1.4 S9R, R10K, P14Q, T22A, Y167A, R170S 1.6 S9R, *22aL,S57A, G61E, *98aA, V139L, N173S 1.3 P14T, N18K, Y167A, R170S 2.0 S9R,Q12E, P14Q, K27R, Y167A, R170S 1.7 N62D, R170L 1.7 N62D, R170S, Q245R1.4 Y167A, R170S, A194P, K251R, S265K 2.0 P14T, N18K, Y167A, R170S,A194P 2.0 N62D, A151G, K237R 2.0 N62D, A151G, Q245R 1.9 N62D, A151G,K237R, Q245R 2.0

EXAMPLE 4

Wash Performance of Detergent Composition Comprising Subtilase Variants

The milliliter scale wash performance assay was conducted under thefollowing conditions:

Mini Wash Assay Detergent Persil, Lever, UK, HDP Detergent dose 6 g/l pHAs it is Wash time 20 min. Temperature 30° C. Water hardness 15°dH,adjusted by adding CaCl₂*2H₂O, MgCl₂*6H₂O and NaHCO₃ (4:1:7.5) tomilli-Q water. Enzyme conc. 2.5 nM, 5 nM, 10 nM, 30 nM, 60 nM Testsystem 125 ml glass beakers. Textile dipped in test solution.Continuously lifted up and down into the detergent solution, 50 timesper minute. Swatch used: EMPA 116 (2.5 cm × 7 cm) Test solution volume50 ml

After washing the textile piece is flushed in tap water and air-driedand the remission (R) of the test material is measured at 460 nm using aZeiss MCS 521 VIS spectrophotometer. The measurements are done accordingto the manufacturer's protocol.

The performance of the new variants is compared to the performance ofSavinase at 10 nM protease concentration by calculating the relativeperformance:RP=(R _(variant) −R _(BLANK))/(R _(SAVINASE) −R _(BLANK))

A variant is considered to exhibit improved wash performance, if itperforms better than the reference in at least one detergentcomposition.

Scoring: A score=1 is given for variants with an improved washperformance equal to or better than 1.1. Mutations Score S103A, V104I,G159D, A232V, Q236H, Q245R 1 S9R, A15T, T22A, V139L 1 S9R, A15T, G61E,A85T, E89Q, P239L, Q245C 1 S9R, A15T, V68A, H120N, Q245R 1 N248R 1 S9R,A15T, *22aL, V139L, N204D, Q245L 1 N218S 1 S9R, A15T, V68A, Q245R, N252K1 S9R, A15T, V68A, Q245R, H120N 1 V68A, S106A, H120N 1 V68A, S106A,N252K 1 A15T, V68A, S99G, Q245R, N261D 1 S9R, V68A, S99G, Q245R, N261D 1V68A, S99G, Q245R, N261D 1 S9R, A15T, V68A, S99G, N261D 1 S9R, A15T,V68A, Q245R, N261D 1 S9R, A15T, *22aL, V139L, S163G, N204D, Q245L 1Q245R, N252H 1 S9R, *22aL, G61E, *97aA, M119I, Q137H, N173S 1 V68A,S106A, T213A 1 S9R, A15T, V68A, H120N, P131S, Q137H, Q245M 1 S9R, A15T,V68A, I72F, S99G, Q245R, N261D 1 S9R, A15T, V68A, S99D, Q245R, N261D 1S9R, A15T, V68A, S99G, A194P, Q245R, N261D 1 S9R, A15T, V68A, N76I,S99G, Q245R, N261D 1 S9R, A15T, V68A, S99G, A228V, Q245R, N261D 1

1. A subtilase variant comprising one or more sets of modificationsselected from the group consisting of T143K, Y167A, R170S, A194P; Y167A,R170S, A194P, K251 R; Y167A, R170S, A194P, S265K; Y167A, R170S, A194P,V244R; S141E, Y167A, R170S, A194P; Y167A, R170S, M1751; Y167A, R170S,A172T; Y167A, R170S, A174V, M175F; Y167A, R170S, A172V, A174V; Y167A,R170S, A172E; Y167A, R170S, M175L; Y167A, R170S, A174T; Y167A, R170S,A174T, M175L; G53C, G61E; A98S, S99D, G100S; S9R, T22A, V68A, S99A,*99aD; S9R, P14H, R19L, N62D; G61P, *99aS; N43S, N62D; *96aG, P131S,V203A, A228T; N62D, A232C, Q236L, Q245N; *96aA, A98T, R247K; S99D,S101R, S103A, V104I, G160S, A194P, L217D; *61aD; N62D, S106A; V68A,S106M, N184D; S9R, A15T, *97aV, H120N; A15M, A16P, *99aD; *99aE, G160S,S163T, G195S, G211S, K237R, G258A, T260L; G23S, *99aD, A194P, S242T,Q245R; G100S, N173D; Y167A, R170S, A172E; A98T, Q137L, Y167A, R170S,M175L; *98aA, S99D; S99A, *99aD, V203A; N62D, K237R; V11M, N76D, L126F,K251R; S9F, A15L, A16P, T22I, *98aA, S99D, R170H; *96aA, *130aG, P131H;E54D, N62D; *98aA, *98bS, S99G, S101T; S9R, A15T, V68A, I79T, G102S,P131H, Q137H; *100aA, *100bG, *100cS, *100dG; V68A, L111I; *98aA, R170H,Q245R; I35V, N62D, N183D, T224S; *97aG, P131S, V203A, A228T; S9R, R10K,P14Q, T22A, Y167A, R170S; S9R, *22aL, S57A, G61E, *98aA, V139L, N173S;P14T, N18K, Y167A, R170S; S9R, Q12E, P14Q, K27R, Y167A, R170S; N62D,R170L; N62D, R170S, Q245R; Y167A, R170S, A194P, K251R, S265K; P14T,N18K, Y167A, R170S, A194P; N62D, A151G, K237R; N62D, A151G, Q245R; N62D,A151G, K237R, Q245R; S103A, V104I, G159D, A232V, Q236H, Q245R; S9R,A15T, T22A, V139L; S9R, A15T, G61E, A85T, E89Q, P239L, Q245C; S9R, A15T,V68A, H120N, Q245R; N248R; S9R, A15T, *22aL, V139L, N204D, Q245L; N218S;S9R, A15T, V68A, Q245R, N252K; S9R, A15T, V68A, Q245R, H120N; V68A,S106A, H120N; V68A, S106A, N252K; A15T, V68A, S99G, Q245R, N261D; S9R,V68A, S99G, Q245R, N261D; V68A, S99G, Q245R, N261D; S9R, A15T, V68A,S99G, N261D; S9R, A15T, V68A, Q245R, N261D; S9R, A15T, *22aL, V139L,S163G, N204D, Q245L; Q245R, N252H; S9R, *22aL, G61 E, *97aA, M119I,Q137H, N173S; V68A, S106A, T213A; S9R, A15T, V68A, H₁₂₀N, P131S, Q137H,Q245M; S9R, A15T, V68A, 172F, S99G, Q245R, N261D; S9R, A15T, V68A, S99D,Q245R, N261D; S9R, A15T, V68A, S99G, A194P, Q245R, N261D; S9R, A15T,V68A, N76I, S99G, Q245R, N261D and S9R, A15T, V68A, S99G, A228V, Q245R,N261D.
 2. A subtilase variant according to claim 1, wherein said variantfurther comprises one or more of the modifications K27R, *36D, S56P,N62D, V68A, N76D, S87N, G97N, S99SE, S101G, S101R, S103A, V104A, V104I,V104N, V104Y, S106A, H120D, H120N, N123S, G159D, Y167A, R170S, R170L,A194P, N204D, V2051, Q206E, L217D, N218S, N218D, M222S, M222A, T224S,A232V, K235L, Q236H, Q245R, N248D, N252K, T274A, S101G+V104N,S87N+S100G+V104N, K27R+V104Y+N123S+T274A, N76D+S103A+V104I,S99D+S101R+S103A+V104I+G160S,S3T+V4I+S99D+S101R+S103A+V104I+G160S+V199M+V205I+L217D,S3T+V4I+S99D+S101R+S103A+V104I+G160S+A194P+V199M+V205I+L217D,S3T+V4I+S99D+S101R+S103A+V104I+G160S+V205I and N76D+V104A.
 3. Asubtilase variant according to claim 1 comprising the followingsubstitutions: S101 G+S103A+V104I+G159D+A232V+Q236H+Q245R+N248D+N252K.4. The variant according to claim 1, wherein the parent subtilasebelongs to the sub-group I-S1.
 5. The variant according to claim 1,wherein the parent subtilase belongs to the sub-group I-S2, and whereinthe parent preferably is BLSAVI.
 6. A cleaning or detergent composition,comprising a variant of claim 1 and a surfactant.
 7. A composition ofclaim 6, which additionally comprises a cellulase, a lipase, an amylase,a cutinase, a protease, a hemicellulase, an esterase, a lactase, aglycoamylase, a polygalacturonase, a beta-galactosidase, a ligninase, ora mixture thereof.
 8. An isolated DNA sequence encoding a subtilasevariant of claim
 1. 9. An expression vector comprising the isolated DNAsequence of claim
 8. 10. A microbial host cell transformed with theexpression vector of claim
 9. 11. A microbial host cell of claim 10,which is a bacterium.
 12. A microbial host cell of claim 10, which is afungus or yeast.
 13. A method for producing a subtilase variant,comprising (a) culturing a host of claim 1 under conditions conducive tothe expression and secretion of the variant, and (b) recovering thevariant.